Improved methodologies for maximizing protein production through recombinant gene expression is an on-going effort in the art of particular interest is the development of methodologies for maximizing the folding of polypeptide chains produced by recombinant gene expression as a way of producing commercially useful quantities of functional proteins.
A variety of eukaryotic vectors and eukaryotic host cell systems have been developed for the production of proteins by recombinant gene expression. While eukaryotic cells, typically yeast's and mammalian cell lines, have been used in the production of a variety of mammalian proteins, a number of problems associated with such cells still exist. Specifically, high levels of protein production typically are not easily obtained in eukaryotic expression systems. In addition, eukaryotic host cells typically have stringent growth requirements and have slow growth rates in culture. Consequently, the production of large quantities of recombinant proteins requires more than simply culturing a host cell transfected with an expression vector. This is particularly true when the gene of interest encodes a protein that is poorly expressed, e.g., is not produced in abundance and/or is only transiently produced under natural, physiological conditions.
In contrast, prokaryotic host cell systems, typically bacterial host cell systems, have proven capable of generating large quantities of proteins by recombinant gene expression. Typically, bacterial host cells have much simpler growth requirements and grow significantly faster than eukaryotic cells thereby enabling the production of large quantities of recombinant proteins at a fraction of the cost of producing equivalent quantities of the same protein in a eukaryotic system. To date, the prokaryotic organism Escherichia coli (E. coli), typically has been the organism of choice. A variety of E. coli expression vectors, E. coli host strains and methods for expressing recombinant proteins in E. coli are well known, and thoroughly discussed in the art. See, for example, Rosenberg et al. (1983) Methods in Enzymology 101:123; Milman (1987) Methods in Enzymology 153:; Duffaud et al. (1987) Methods in Enzymology 153:; Sambrook et al. ed., (1989), Molecular Cloning: A Laboratory Manual, 2nd Edit., Cold Spring Harbor Laboratory Press; Ausubel et al., ed., (1989) Current Protocols in Molecular Biology, John Wiley & Sons, NY, the disclosures of which are incorporated herein by reference.
High levels of protein production by recombinant gene expression in E. coli, typically results in the formation of insoluble aggregates of the unfolded polypeptide in the E. coli cytoplasm. The insoluble aggregates, known as "inclusion bodies", may be purified by methodologies well known in the art, and described for example, in Sambrook et al., (1989) supra. Following expression of the protein of interest, inclusion bodies are isolated, washed, and the insoluble inclusion bodies solubilized in buffer. Useful solubilization agents include for example, chaotropic salt and detergents. A number of strategies useful in identifying effective solubilization agents are described in Glover (1987) DNA Cloning: A Practical Approach, IRL Press, Oxford, the disclosure of which is incorporated herein by reference.
The solubilized polypeptides subsequently are folded in vitro to form a biologically active protein. Folding is initiated by diluting the solubilization reagent, however, optimal folding conditions typically are found by empirical experimentation. Folding of proteins containing cysteine residues has proved difficult because incorrect intramolecular and intermolecular disulfide bonds may form during folding preventing the polypeptide from attaining its biologically active conformation. To date, only a few functional mammalian proteins, i.e., growth hormone and synthetic hemoglobin, have been produced following their expression E. coli. Accordingly, the development of procedures for folding polypeptides produced by recombinant gene expression in E. coli is an on-going effort in the art.
Although the mechanisms by which polypeptide chains fold into their native conformations are poorly understood, it is believed that folding of a polypeptide chain is initiated by the collapse of hydrophobic domains into the interior of the molecule, formation of stable secondary structures; and/or the formation of covalent interactions, i.e., disulfide bridges, which stabilize the polypeptide in a particular conformation. Subsequent folding appears to occur through a limited number of pathways involving distinct intermediates. These intermediates appear to be in rapid equilibrium with the denatured state and are only converted slowly to the native biologically active state (Gething et al., supra).
Although some polypeptides have the ability to assemble spontaneously into their native biologically active conformation in vitro many commercially useful proteins do not. There appear to be several competing processes which together influence whether a polypeptide chain will fold spontaneously and include, for example, the formation of unfavorable intramolecular and/or intermolecular interactions that together result in the aggregation of the polypeptide chains. It is believed that the aggregated polypeptide chains may no longer participate in a productive folding pathway.
It has been found, however, that a family of extrinsic helper proteins, called chaperonins, may prevent unfavorable interactions between unfolded polypeptide chains thereby assisting the polypeptides to fold along productive folding pathways. Chaperonins are oligomeric protein complexes that mediate the correct assembly of a preselected polypeptide chain, but which are not themselves components of the final functional protein. Chaperonins not only promote the folding of monomeric proteins but also oligomeric protein complexes. The roles of chaperonins in protein folding have been reviewed extensively. See, for example: Zeilstra-Ryalls et al. (1991) Annu. Rev. Microbiol. 45:301-325; Hartl et al. (1992) Annu. Rev. Biophys. Biomol. Struct. 21:293-322; Lorimer (1992) Curr. Opin. Struct. Biol. 2:26-34; Gething et al. (1992) Nature 322:33-45; and Ellis et al. (1991) Annu. Rev. Biochem. 60:321-347, the disclosures of which are incorporated herein by reference.
The term chaperonin originally was used to define a class of molecular chaperones homologous in structure to E. coli GroEL. The earliest evidence for the involvement of the chaperonins in post-translational events was the demonstration that E. coli groEL plays an integral role in phage head development (Sternberg et al. (1973) J. Mol. Biol. 76:25-44; Georgopoulos et al. (1973) J. Mol. Biol. 76:45-60). Members of this protein family, however, are present in all prokaryotes and in those organelles of eukaryotic cells, such as mitochondria (also known as heat shock protein-60) and chloroplasts (also known as ribulose-1,5-bisphosphate carboxylase subunit binding protein), that have probable endosymbiotic origin. These proteins, which have been renamed chaperonin-60 (cpn60), typically are large tetradecameric complexes composed of 14 identical non covalently associated subunits arranged as two stacked heptameric toroidal rings having intrinsic ATPase activity (Hohn et al. (1979) J. Mol. Biol. 129:359-373; Hendrix (1979) J. Mol. Biol. 129:375-392; Puskin et al. (1982) Biochim. Biophys. Acta 704:379-384; Ishii et al. (1992) FEBS Let. 299:169-174). It has been shown, however, that a functional mammalian mitochondrial cpn60 complex may exist as a single heptameric toroidal ring (Viitanen et al. (1992) J. Biol. Chem. 267:695-698).
Each subunit of the cpn60 complex has an apparent relative molecular weight of about 60 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 60 kD monomeric subunits are referred to herein as monomeric subunits of cpn60 or cpn60m. Lissin et al. (1990) Nature 348:339-342 show that the in vitro assembly of oligomeric E. coli cpn60 complex from monomeric subunits of cpn60 requires Mg.sup.2+ and ATP.
Furthermore, it has been found that in the presence of Mg.sup.2+ and ATP the cpn60 complex may associate with a co-chaperonin called cpn10 to form holo-chaperonin. Native cpn10 is believed to exist as a oligomeric complex comprising seven identical subunits each of which have an apparent molecular weight of about 10 kD as determined by SDS-PAGE (Chandrasekhar et al. (1986) J. Biol. Chem. 261:1214-12419; Lubben et al. (1990) Proc. Natl. Acad. Sci. USA 87:7683-7687; Hartman et al. (1992) Proc. Natl. Acad. Sci. USA 89:3394). Like cpn60 complexes, the cpn10 complexes have been found in both prokaryotes and eukaryotes. For example, a prokaryotic cpn10 complex, called GroES, has been isolated from E. coli and a eukaryotic cpn10 homologue has been isolated from mitochondria.
Presently, the functional bacterial chaperonin complex or holo-chaperonin is believed to comprise the tetradecameric form of cpn60 and the heptameric form of cpn10. It is believed also that the binding of heptameric cpn10 to tetradecameric cpn60 regulates the ATPase activity of the cpn60 complex, and further, that cpn10 is required optimal activity of the cpn60 complex in protein folding (Chandrasekhar (1986), supra; Viitanen (1990) Biochem. 29:5665-5671; Martin et al. (1991) Nature 352:36-42).
Studies have shown that the amino acid sequences of the monomeric subunits of cpn60 and cpn10, are highly conserved between prokaryotes and eukaryotes (Hemmingsen et al. (1988) Nature 333:330-334; Zeilstra-Ryalls (1991), supra, Ellis et al. (1991), supra). For example, yeast mitochondrial chaperonin 60 protein shares 45% identical residues with wheat plastid chaperonin 60, and 54% identical residues with E. coli chaperonin 60 (Ellis et al. (1991), supra).
In vitro studies show that the tetradecameric form of chaperonin-60 typically binds unfolded polypeptide chains thereby arresting their folding. It is believed that heptameric cpn10 coordinates conformational changes of in the cpn60 complex thereby allowing the folding of the polypeptide chain to occur by a process of stepwise release (Martin et al., (1991) supra). Structural and biochemical studies have shown that the binding site for the unfolded polypeptide chain is in the central cavity of the tetradecameric cpn60 complex (Langer et al. (1992) EMBO J. 11:4757-4765; Braig et al. (1993) Proc. Natl. Acad. Sci. USA. 90:3978-3982).
Goloubinoff et al. (1989) Nature 324:884-889 show that the oligomeric chaperonin-60 complex is required for the generation of a functional holo-chaperonin complex. Heretofore, it was believed that only functional holo-chaperonin complexes comprising oligomeric chaperonin-60 and heptameric chaperonin-10 are capable of promoting the folding of polypeptide chains in vitro. Under certain experimental conditions, for example, in the presence of chaotropic agents it may be difficult to maintain the stability of functional holo-chaperonin complex. Accordingly, it is desirable to develop protein compositions which promote the folding of polypeptide chains in vitro irrespective of whether or not the proteins are functionally active as monomers or oligomers. In addition, it is desirable to develop simpler procedures for promoting the folding of polypeptide chains in vitro.
Accordingly, it is an object of the instant invention to provide a composition comprising monomeric subunits of cpn60, or truncated fragments thereof, which promote the folding of a preselected polypeptide chain in vitro. It is another object of the invention to provide immobilized monomeric subunits of cpn60, or fragments thereof, which promote the folding of a preselected polypeptide chain in vitro. It is another object of the invention to provide methods using the aforementioned compositions for folding preselected polypeptide chains in vitro. Importantly, it is another object of the instant invention to provide a means for producing commercially-feasible quantities of biologically active proteins following their expression in a heterologous, specifically a prokaryotic, expression system.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.